An integrated approach to the study of enzyme mechanism will be developed by directly inspecting reaction intermediates at subzero temperatures under rapid detection conditions. The project will consist of three major phases. The first phase will be the construction of a low temperature stopped-flow instrument which can function at -50 degrees C. It will be designed to be readily interfaced with a variety of light sources, detectors and rapid scanning devices. The second phase will be its testing at subzero termperatures on well studied bioorganic model systems designed to mimic enzymes. These systems will include model transamination reactions and base catalyzed hydrolyses of nitrophenolate esters. The third phase will use seryl and metalloenzymes, particularly peptidases, for which spectral systems have been designed to allow direct visualization of reaction intermediates. These enzyme systems will serve both to explore their particular mechanisms of action and to become the basis for generalizations of the approaches found suitable.